Transcription in bacteria is carried out by a multi-subunit RNA polymerase (RNAP) that is conserved from bacteria to humans. Promoter-specific transcription initiation depends on the ??subunit of RNAP, which must associate with the catalytically proficient core enzyme to form the holoenzyme. Historically defined as an initiation factor, ? can also remain associated with the transcription elongation complex and influence the transcription process during elongation. In particular, ? can induce early elongation pausing by establishing sequence-specific interactions with promoter-like DNA sequence elements within the initial transcribed region of a gene. The proposed research builds on the demonstration that such promoter-like pause- inducing elements can function to inhibit ? loss during the earliest stage of elongation, increasing the ? content of elongation complexes throughout the transcription unit. The results of bioinformatic analyses suggest that promoter-proximal ?-dependent pause elements are associated with at least 20% of all E. coli promoters, which implies that this mechanism for regulating the ? content of elongation complexes is likely a general feature of transcription in E. coli. The first aim of the proposed research is to investigae the effects of ? retention on E. coli gene expression. The second aim is to investigate whether the potential roles for ? during transcription elongation extend beyond the primary ? factors and to determine whether different types of ? factors can participate in initiation and elongation on the same transcription unit (? cross-regulation). The third aim is to investigate the mechanism by which promoter-proximal pause elements influence the ? content of downstream elongation complexes. The fourth aim is to investigate whether a specific ?-core interaction that may be essential for cell viability facilitates the release of ? from the DNA in the context of a paused early elongation complex. Together, the proposed experiments will lead to a deeper understanding of the roles of a classical initiation factor during transcription elongation, contributing to an accumulating body of evidence for the importance of post-initiation regulatory events in both prokaryotes and eukaryotes. PUBLIC HEALTH RELEVANCE: RNA polymerase (RNAP), which is responsible for the process of transcription, is the central enzyme of gene expression. An important antimicrobial target, the bacterial RNAP has provided a crucial model for understanding transcription, and the principles that emerge from these investigations continue to inform the study of the many transcription-based processes that underlie human development and human disease. The proposed research is focused on an essential component of RNAP, the historically defined transcription initiation factor ?, which is emerging as a general transcription factor that influences all stages of the transcription cycle.